JPWO2007023711A1 - Microbial culture apparatus, culture vessel, and culture method - Google Patents

Abstract

A culture medium tank 11 in which the culture medium F is placed, a partition plate 12 disposed on the upper end side of the culture medium tank 11, a number of incubators 14 arranged upright on both upper and lower sides of the partition plate 12, and culture The isolation culture apparatus 10 is configured to include a gas supply means 17 disposed inside the liquid tank 11. Each incubator 14 includes a culture unit 19 that is located below the partition plate 12 and is immersed in the culture solution F in the culture solution tank 11, and an injection unit 20 that is located above the partition plate 12. Yes. The culture unit 19 is composed of a film body surrounding the target microorganism, and this film body is formed so as to allow passage of various chemical substances while preventing passage of various microorganisms. By incorporating F into the membrane, the culture environment of the target microorganism is made substantially the same as the culture environment outside the membrane.

Description

  The present invention relates to a microorganism culture apparatus, culture apparatus, and culture method, and more particularly to a microorganism culture apparatus, culture apparatus, and culture method suitable for isolating and culturing more types of microorganisms.

  A wide variety of microorganisms exist in the natural environment, but these microorganisms form a complex microbial system, and more microorganisms are isolated from this complex microbial system and cultured after the isolation. By doing so, it becomes possible to find useful microorganisms or use them as unused gene resources.

  By the way, as a conventional method for isolation and culture of microorganisms, there is a plate culture method introduced in Patent Document 1. In this plate culture method, the sample containing the target microorganism is diluted to reduce the number of cells per predetermined unit of the target microorganism, and then the diluted sample is placed on the agar medium in the container, This is a method for culturing and proliferating a target microorganism over a period of time.

  Furthermore, a liquid limiting dilution culture method is known as a technique for culturing microorganisms (see, for example, Patent Document 2), and this method is also used as a general technique for isolating and culturing microorganisms. In the isolation and culture of microorganisms performed by this liquid limit dilution method, containers such as a large number of test tubes are used, a predetermined liquid medium is added to each container, and 0 or 1 cell of the target microorganism is contained in a predetermined solution. Dispense the diluted product into each container and culture. As a result, a single type of microorganism can be separated and cultured.

  As another isolation culture method, as disclosed in Patent Document 3, a method using a hermetic container having a membrane that does not allow microorganisms to pass but allows only water to pass therethrough is known. In this method, an agar medium inoculated with a bacterial source containing microorganisms to be separated is enclosed in the hermetic container, and then the hermetic container is placed in an external environment according to the bacterial source. At this time, an organic substance having a low concentration is supplied from the external environment to the agar medium through the membrane, and various microorganisms in the agar medium are grown to form colonies of the various microorganisms. A colony of microorganisms to be separated is extracted from them.

Furthermore, as disclosed in Patent Document 4, there is also a method for isolating and culturing microorganisms using an electric culture apparatus. The electroculturing apparatus here is an apparatus for culturing microorganisms while applying a voltage to a medium. The electric culture apparatus includes a first electrode, a plurality of culture tanks penetrating the first electrode, a counter electrode tank connected to the culture tank, a second electrode disposed in the counter electrode tank, and the culture An ion exchange membrane and a filter for partitioning the tank and the counter electrode tank, and a gas supply means for supplying a gas containing an element necessary for culturing microorganisms to the counter electrode tank. The culture tank is filled with microorganisms suspended in a medium containing iron oxide, and the counter electrode tank is filled with a solution containing medium components not containing iron. The filter prevents movement of microorganisms between the culture tank and the counter electrode tank. With the above configuration, when a voltage is applied to the first and second electrodes, the hydrogen and carbon supplied into the counter electrode tank by the gas supply means pass through the filter from the medium in the counter electrode tank and pass through the filter. By moving to a medium and having an ionic reaction with the iron component in the medium, the microorganisms therein are electrically isolated and cultured. At this time, iron ions are prevented from flowing out to the counter electrode tank by the ion exchange membrane, and are repeatedly used in the culture tank.
JP-A-6-277095 JP-A-11-253153 JP-A-1-265882 JP 2004-16023 A

  However, the conventional culture methods described above cannot isolate and culture most microorganisms on the earth. This is considered to be because each conventional culture method is performed in an environment far from the actual environment where microorganisms inhabit. This will be described in detail below.

  In the plate culture method and the liquid limit dilution culture method, microorganisms that are not affected by the concentration of the substrate, the presence of metabolites, etc. can be isolated and cultured, but there are few such microorganisms. In other words, in the related art, since the culture is performed in a closed environment where excess substrate and microbial product cannot be discharged, the metabolite produced by the microorganism to be cultured increases in concentration over time. And signal factors accumulate, microorganisms that are inhibited by them cannot grow.

  In addition, in the plate culture method and the liquid limiting dilution culture method, unlike the actual environment, the microorganisms to be cultured are cultured alone, so that the microorganisms proliferate upon interaction with the surrounding symbiotic microorganisms. It is not suitable for culturing. Here, it is also conceivable to add a symbiotic microorganism to the target microorganism and culture, but in the isolation culture operation, the physiological ecology of the target microorganism is unknown, and the presence or type of the symbiotic microorganism is unknown. It is impossible except for accidental contamination of symbiotic microorganisms.

  In the plate culture method and the liquid limit dilution culture method, the pH and residual oxygen concentration of the culture environment cannot be adjusted during the culture, and microorganisms sensitive to these pH and residual oxygen concentration cannot grow.

  In the method disclosed in Patent Document 3, the inside of the membrane is sealed in a solid medium composed of an agar medium or the like inoculated with a bacterial source, while the outside of the membrane is an external environment according to the bacterial source. Therefore, the culture environment differs between the inside and outside of the membrane. As a result, the diffusion rate of substances such as organic matter between the inside and outside of the membrane is surely slower than the environment in which microbial cells are actually cultured, and the environment inside the membrane is sensitive to changes in the external environment in a short time. I can't respond. Since some microorganisms affect the culture, this method also has a limit in isolating and culturing more types of microorganisms.

  Further, in the method of Patent Document 3, colonies must be formed on an agar medium and colonies must be extracted by a known agar overlay method or the like. There is also a problem that work takes time and effort. In addition, it is known that there are many microorganisms that do not form colonies in nature or that form only microcolonies that are so small that they cannot be observed with the naked eye. It is difficult to carry out separation culture.

  In the method disclosed in Patent Document 4 for electroculturing microorganisms, microorganisms that can be cultured by electroculture are limited to only a few types in the environment, such as iron-reducing bacteria, and cannot be applied to many environmental microorganisms in general. . That is, in the apparatus of Patent Document 4, the components of the medium to be put in the culture tank and the counter electrode tank are artificially different so as to be suitable for electrocultivation, and artificially separated from the natural state in which microorganisms grow. Only a limited number of microorganisms can be isolated and cultured for culturing.

  The present invention has been devised by paying attention to the above problems, and its purpose is that microorganisms can be isolated and cultured in an environment close to the actual environment where microorganisms inhabit, and more types of microorganisms can be obtained. It is an object of the present invention to provide a microorganism culture apparatus and incubator, and a culture method that enable isolation and culture of the microorganism.

(1) In order to achieve the above object, an isolation culture apparatus of the present invention includes a culture solution tank in which a culture solution is placed, and a culture unit in which a target microorganism is cultured by being immersed in the culture solution in the culture solution tank. And
The culture unit is configured by a membrane body surrounding the target microorganism, and the membrane body is formed to allow passage of various chemical substances while preventing passage of various microorganisms, and culture in the culture solution tank. By taking the liquid into the membrane, the culture environment of the target microorganism is made substantially the same as the culture environment outside the membrane.

(2) Moreover, the isolation culture apparatus of the present invention includes a culture solution tank in which a culture solution is placed, and a culture unit in which the target microorganism is cultured by being immersed in the culture solution in the culture solution tank,
The culture part is constituted by a membrane body surrounding the target microorganism, and the membrane body has a hole having an inner diameter of 1 μm or less, and takes the culture solution in the culture solution tank into the membrane body, thereby the target microorganism. It is also possible to adopt a configuration in which the culture environment is substantially the same as the culture environment outside the membrane body.

  (3) In the above, it is preferable that the membrane body is composed of a porous hollow fiber membrane having an inner diameter of 5 mm or less.

  (4) Moreover, the structure that the gas supply means which supplies predetermined | prescribed gas in the said culture solution is provided in the said culture solution tank can also be employ | adopted collectively.

  (5) Furthermore, it is good to provide the injection | pouring means which inject | pours the said target microorganisms into the said culture part.

(6) Further, the incubator of the present invention comprises a culture part where the target microorganism is cultured by being immersed in a predetermined culture solution, and an injection means for injecting the target microorganism into the culture part,
The culture unit is constituted by a membrane body surrounding the target microorganism, and the membrane body is formed to allow passage of various chemical substances while preventing passage of various microorganisms, and the culture solution is passed through the membrane body. In other words, the culture environment of the target microorganism is made substantially the same as the culture environment outside the membrane body.

  (7) Further, the isolation culture method of the present invention prevents the passage of various microorganisms, while surrounding the target microorganism with a membrane body that allows passage of various chemical substances, A technique is employed in which the target microorganism is cultured in a culture environment substantially the same as the culture environment outside the membrane body by dipping in the culture solution and taking the culture solution into the membrane body.

(8) A method for culturing microorganisms using a membrane that prevents passage of various microorganisms while allowing passage of various chemical substances,
A sample containing the target microorganism is diluted, the sample containing at least one target microorganism is surrounded by the film body, the film body is immersed in a predetermined culture solution, and the culture solution is taken into the film body. Thus, it is possible to adopt a technique of culturing the target microorganism in a culture environment almost the same as the culture environment outside the membrane body.

  According to the present invention, a group of microorganisms present in the actual growth environment of the target microorganism, such as seawater or wastewater in a wastewater treatment apparatus, is outside the target microorganism surrounded by a film body that passes a chemical substance without passing through the microorganism. There is a miscellaneous culture solution, and since the culture solution is taken into the membrane, microorganisms can be isolated and cultured in the membrane in a state close to the actual environment where the target microorganisms inhabit, Many types of microorganisms can be isolated and cultured.

  That is, in the culture solution, the target microorganism is cultured in a state surrounded by the above-mentioned membrane body, so that metabolites and signal factors produced by itself can be naturally discharged into the culture solution outside the membrane, When the target microorganism is grown, the influence of the metabolite and signal factor can be reduced.

  Furthermore, while the signal factors of other microorganisms present in the culture solution outside the membrane can be taken into the membrane, the other microorganisms do not enter the membrane, and thus other microorganisms useful for the growth of the target microorganism. While taking in the signal factor, complexation with other microorganisms can be avoided, and the types of target microorganisms that can be grown can be increased.

  In addition, since the culture solution can be adjusted as an open system outside the membrane body, the concentration of the substrate in the culture solution, the pH in the solution, and the dissolved oxygen concentration can be easily adjusted according to the state of the actual environment. It becomes possible to isolate and cultivate microorganisms that are sensitive to heat.

  Furthermore, since the culture medium is taken into the membrane, the culture environment of the target microorganism becomes a liquid state that is almost the same as the culture environment outside the membrane body. Therefore, the conventional method using a fixed medium such as an agar medium is used. On the other hand, the diffusion rate of the substance between the inside and outside of the membrane can be greatly improved, the inside of the membrane can be responded sensitively and in a short time to changes in the external environment, and culture in a state close to the natural state becomes possible.

  Further, in the present invention, without using a fixed medium such as an agar medium, it is possible to isolate and culture a sample containing one target microorganism by diluting a sample containing the target microorganism, It is no longer necessary to extract colonies of target microorganisms from various microorganisms, and microorganisms can be isolated and cultured with simple operations. Also, for microorganisms that do not form colonies and those that form microcolonys. Isolation culture becomes possible.

  In particular, according to the configuration (3), the diffusion rate of the substance between the inside and outside of the membrane can be further improved. In addition, when a sample containing the target microorganism is diluted and a sample containing one target microorganism is put into the membrane and isolated and cultured, an enormous number of culture sections are required. Even so, the culture part can be integrated to make the whole compact.

  Moreover, by configuring as in the above (4), it becomes easy to control the gas supply state in the culture solution more closely to the actual environment.

  Furthermore, according to the configuration of (5), it is possible to easily inject microorganisms into the membrane body, which is useful when a large number of culture sections are prepared.

  Hereinafter, embodiments of the present invention will be described with reference to the drawings.

  FIG. 1 shows a schematic cross-sectional front view of a microorganism culturing apparatus according to the present embodiment. In this figure, a culture apparatus 10 includes a culture solution tank 11 into which a culture solution F is placed, a partition plate 12 that is detachably attached to the upper end side of the culture solution tank 11 and closes the culture solution tank 11 from above. A number of incubators 14 that are supported by the partition plate 12 and are erected on both upper and lower sides across the partition plate 12, a sealed case 15 that is detachably disposed above the partition plate 12, and a culture solution tank 11 And a gas supply means 17 arranged inside.

  The culture solution F to be put into the culture solution tank 11 is various solutions in the environment where microorganisms exist. For example, in a natural environment such as seawater, lake water, river water, or in an environmental purification process such as wastewater treatment. Examples include solutions in an engineering environment such as waste water and treated water.

  Although the said incubator 14 is not specifically limited, About 50-100 sets are provided. As shown in FIGS. 1 and 2, each of these incubators 14 is located below the partition plate 12 and is immersed in the culture solution F in the culture solution tank 11. It is comprised by the injection | pouring part 20 as an injection | pouring means located in the upper side.

  The culture part 19 has a tube shape in which both end sides in the extending direction are open, and specifically, is constituted by a porous hollow fiber membrane (membrane body) having hydrophilicity, in the internal space of the culture part 19 A microorganism to be cultured (hereinafter referred to as “target microorganism”) is placed. Although the culture part 19 is not specifically limited, it is formed of a polysulfone film or a PVDF (polyvinylidene fluoride) film, and has an inner diameter of 0.7 mm, a length of 20 cm to 30 cm, and a pore diameter of 0.1 μm. The inner diameter of the culture unit 19 is preferably 5 mm or less, more preferably 1 mm or less in consideration of space saving of the apparatus and the diffusion rate of the substance between the inside and outside of the membrane.

  Note that the culture unit 19 may be constituted by another film body that can enclose and confine the target microorganism. As this film body, as schematically shown in FIG. Various substances can be adopted as long as the pores H that allow passage of chemical substances including various substrates, metabolites, ions and signal factors are formed while blocking the passage of the microorganism B, Specifically, the pore diameter is preferably 1 μm or less, more preferably about 0.1 μm.

  As shown in FIG. 2, the injection part 20 includes an injection container 22 on the right side in the figure connected to one end side of each culture part 19 and a syringe 23 on the left side in the figure connected to the other end side of each culture part 19. It is comprised by.

  The upper and lower ends of the injection container 22 are open, and the open portion on the upper end side serves as an injection port 22A for the sample solution containing the target microorganism, while the open portion on the lower end side has no gap on one end side of the culture unit 19. It becomes the connection port 22B connected.

  The syringe 23 includes a cylindrical main body 25 and a piston 26 that slides in the main body 25 in the vertical direction. An opening 25A is provided on the lower end side of the main body 25, and the other end side of the culture unit 19 is connected to the opening 25A without a gap. For this reason, the inner space of the injection container 22 and the syringe 23 communicates through the inner space of the culture unit 19.

  As shown in FIG. 1, the sealing case 15 is disposed so as to cover and seal all the injection parts 20 that are erected on the partition plate 12, and from the outside of the sealing case 15 to the injection part 20. It is provided so that the invasion of bacterial cells can be regulated.

  The gas supply means 17 uses a known device capable of supplying a gas such as air or nitrogen gas to the culture solution F in the culture solution tank 11 at a predetermined timing. Since it is not the essence of the invention, the description is omitted here.

  Next, a method for isolating and culturing microorganisms using the above isolation culture apparatus 10 will be described.

  First, dispersion and dilution are performed on sample solution collected from seawater or wastewater of a phosphorus removal reactor as a wastewater treatment apparatus. Specifically, after dispersing cells (aggregates) aggregated in the sample solution one by one with an ultrasonic dispersion device, several types of filters with different eye roughness (for example, pore diameters of 25 μm and 3 μm) are used. Two types) are used to remove dust and aggregates that are not completely dispersed.

  The number of cells (cell / ml) in the sample solution is determined by measuring the total number of cells in the sample solution treated in this way by the DAPI staining direct counting method.

  Then, this sample solution is diluted with a predetermined dilution solution. That is, here, the sample solution is diluted to a concentration of 0.1 to 1 cell / container so that one or more cells do not enter when the sample solution is injected into the culture unit 19. As the diluted solution, a solution in an environment close to the collected sample solution is used. For example, when the sample solution is seawater, in order to reduce damage to the cells due to changes in osmotic pressure or the like, it is preferable to use a solution that has been subjected to autoclave sterilization after filtering and sterilizing seawater without using seawater as it is. On the other hand, when the sample solution is waste water of a phosphorus removal reactor, a solution having the same composition as the waste water (substrate) flowing into the phosphorus removal reactor may be used.

  Then, the following set is performed in the clean bench. First, the diluted sample solution is injected into each incubator 14. At this time, the diluted sample solution is injected into each of the incubators 14 from the injection port 22A of the injection container 22, and the piston 26 of the corresponding syringe 23 is pulled upward, so that the sample solution in the injection container 22 is obtained. It is sucked in the direction of the culture unit 19 and introduced into each culture unit 19 in each culture vessel 14. When the sample solution has been injected into all the incubators 14, the surface of the partition plate 12 and the entire inner surface of the sealed case 15 are sterilized with ethanol, and the entire injected part 20 is covered with the sealed case 15.

  Furthermore, when the sample solution is seawater, microorganisms are cultured as follows. That is, the seawater and sea mud constituting the culture solution F are put into the culture solution tank 11 and the culture unit 19 of the incubator 14 is immersed in the culture solution F by the gas supply means 17. Air is supplied into the culture medium F. The culture period is not particularly limited and can be arbitrarily set. For example, a culture period of about one month can be exemplified. Moreover, it is good to replace the seawater in the culture solution tank 11 with a cycle of about once a week.

  On the other hand, when the sample solution is the waste water of the phosphorus removal reactor, the microorganisms are cultured as follows. That is, first, waste water as the culture solution F is flowed into the culture solution tank 11 and the culture unit 19 is immersed in the culture solution F. In this state, the culture in the culture solution tank 11 is performed every predetermined time. The microorganisms are cultured while replacing about half of the liquid F with new waste water (substrate). Specifically, first, in a state where the culture unit 19 is immersed in the waste water in the culture solution tank 11, nitrogen gas is supplied from the gas supply means 17 to the waste water, and the culture is performed for several hours under anaerobic conditions by aeration. Thereafter, air is supplied from the gas supply means 17 into the wastewater, and culturing is performed for several hours under aerobic conditions. Next, the supply of gas from the gas supply means 17 is stopped, sludge is settled in the culture solution tank 11, about half of the supernatant of the solution in the culture solution tank 11 is extracted, and new waste water is removed accordingly. It supplies in the culture solution 11, and repeats the process mentioned above. For example, culturing for about one month while performing these steps 3 cycles per day.

  During the above culture, the culture medium F outside the culture section 19 is taken into each culture section 19 due to the properties of the membranes constituting the culture section 19, and other culture medium F is present in the culture medium F. The microbial signal factor is taken into each culture section 19. On the other hand, the metabolite and signal factor produced by the target microorganism T itself are naturally discharged into the culture solution F outside each culture unit 19. At this time, other microorganisms existing outside the culture unit 19 do not enter the culture units 19.

  After the above culture period is completed, the sample solution in which the microorganisms are cultured in each culture unit 19 is extracted. At this time, by pulling the piston 26 of the syringe 23 of each incubator 14 upward, the sample solution after culture in the culture unit 19 is sucked out into the main body 25 of the syringe 23, and the piston 26 is moved to the main body 25. By extracting from the sample solution, it is possible to take out the sample solution after culturing the microorganism that has moved into the main body 25.

  The present inventors conducted an experiment for isolation and culture of microorganisms using the seawater and the waste water of the phosphorus removal reactor as the culture solution F by the above procedure. That is, each sample solution of each incubator 14 obtained by the above-described procedure was subjected to DAPI staining, and whether or not the bacterial cells were growing under a fluorescence microscope, and further, the grown ones were morphologically Whether it was a pure bacteria state was judged visually. Then, total DNA is extracted from the cells determined to be in a pure bacterium state, a DNA sequence reaction is performed, and the base sequence of 16S rDNA is determined. Bacteria state.

  As a result, in addition to a higher isolation and culture effect than the conventional method, it has become possible to isolate and cultivate microorganisms that could not be isolated and cultured conventionally. This is considered to be due to the following reason based on the present invention.

  First, since the state of the culture solution F in the culture solution tank 11 can be easily changed, the increase in the concentration of the substrate can be suppressed, and the culture with respect to the microorganism in which the high concentration of the substrate becomes a growth inhibiting factor. Is possible.

  Secondly, since the culture unit 19 is composed of the above-described film body, a metabolite produced by the target microorganism T can be discharged to the outside of the culture unit 19, and its growth is inhibited by the metabolite produced by itself. Can be cultured.

  Thirdly, since the culture solution F exists inside and outside the culture unit 19, it can be in a state close to the actual environment where the target microorganism T inhabits, and substances from the symbiotic bacteria necessary for the growth of the target microorganism T are film bodies. Culture of microorganisms that are easily supplied to the target microorganism T through and propagated under the influence of commensal bacteria is also possible. Further, invasion of other microorganisms into the inside of the membrane body is prevented, so that complexation with the other microorganisms can be avoided, and microorganisms can be isolated and cultured. Furthermore, since the target microorganism T is cultured in the same culture solution F as the outside of the culture unit 19 without using a solid medium such as an agar medium, the diffusion rate of substances between the inside and outside of the culture unit 19 is increased as compared with the conventional case. It is possible to respond quickly to changes in the external environment such as changes in the natural environment.

  Fourth, the pH and residual oxygen concentration of the culture medium F can be easily changed in the middle, and microorganisms that are sensitive to changes in the pH and residual oxygen concentration of the surrounding environment can be grown.

  In addition, since colony extraction is not required, isolation and culture can be performed for microorganisms that do not form colonies and microorganisms that form microcolony.

  In the above embodiment, the culture apparatus 10 is used to isolate and culture microorganisms. However, the present invention is not limited to this, and the culture apparatus 10 may be used for other cultures of microorganisms. That is, for example, by using the culture apparatus 10 and putting a plurality of types of microorganisms in each culture section 19, it is of course possible to simultaneously culture various microorganisms.

  The configuration of each part of the apparatus in the present invention is not limited to the illustrated configuration example, and various modifications are possible as long as substantially the same operation is achieved.

The schematic sectional front view of the isolation culture apparatus which concerns on this embodiment. The enlarged front view of an incubator. The conceptual cross section which showed the state in a culture part.

Claims (8)

A culture solution tank in which the culture solution is placed, and a culture unit in which the target microorganism is cultured by being immersed in the culture solution in the culture solution tank,
The culture unit is constituted by a membrane body surrounding the target microorganism, and the membrane body is formed to allow passage of various chemical substances while preventing passage of various microorganisms, and is cultured in the culture solution tank. An apparatus for cultivating microorganisms, wherein a culture environment of the target microorganism is made substantially the same as a culture environment outside the membrane body by taking a liquid into the membrane body. A culture solution tank in which the culture solution is placed, and a culture unit in which the target microorganism is cultured by being immersed in the culture solution in the culture solution tank,
The culture part is configured by a membrane body surrounding the target microorganism, and the membrane body has a hole with an inner diameter of 1 μm or less, and takes the culture solution in the culture solution tank into the membrane body, thereby the target microorganism. A culture apparatus for microorganisms, wherein the culture environment is substantially the same as the culture environment outside the membrane.   3. The microorganism culturing apparatus according to claim 1, wherein the membrane body is composed of a porous hollow fiber membrane having an inner diameter of 5 mm or less.   The microorganism culture apparatus according to claim 1, 2 or 3, wherein a gas supply means for supplying a predetermined gas into the culture solution is provided in the culture solution tank.   The microorganism culturing apparatus according to claim 1, further comprising injection means for injecting the target microorganism into the culture unit. A culture unit that is immersed in a predetermined culture solution to culture the target microorganism, and an injection unit that injects the target microorganism into the culture unit,
The culture unit is constituted by a membrane body surrounding the target microorganism, and the membrane body is formed to allow passage of various chemical substances while preventing passage of various microorganisms, and the culture solution is passed through the membrane body. The incubator makes the culture environment of the target microorganism substantially the same as the culture environment outside the membrane body.   While surrounding the target microorganism with a membrane body that prevents passage of various microorganisms while allowing passage of various chemical substances, the membrane body is immersed in a predetermined culture solution, and the culture solution is immersed in the membrane body. A microorganism culturing method characterized by culturing the target microorganism in a culture environment substantially the same as the culture environment outside the membrane body. A method of culturing microorganisms using a membrane that prevents passage of various microorganisms while allowing passage of various chemical substances,
A sample containing the target microorganism is diluted, the sample containing at least one target microorganism is surrounded by the film body, the film body is immersed in a predetermined culture solution, and the culture solution is taken into the film body. Thus, the method for culturing a microorganism is characterized by culturing the target microorganism in a culture environment substantially the same as the culture environment outside the membrane body.

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